Post-transcriptional RNA processing can regulate gene expression, which is essential for the control of cellular metabolism, growth, and differentiation. Broad long-term objectives of this application are to characterize various RNA processing events in Archaea. The specific aims of this proposal are: Structural and functional characterization of archaeal box C/D sRNPs;In vivo characterization of the production of 2'-O-methylcytidine (Cm) and 2'-O-methyluridine (Um) at positions 34 and 39, respectively, of Haloferax volcanii tRNATrp and study, in vivo, the relationship among these modification reactions, splicing and processing of pre-tRNATrp;Determination of the effect of Cm modification at the wobble position of tRNA on the accuracy of translation in H. volcanii;and Characterization of pseudouridine formation at positions 54 and 55 in archaeal tRNAs. Structural and functional studies of dual guide-target RNAs will be done by using recombinant box C/D RNP core proteins along with 32P-labeled T7 RNA polymerase generated pre-tRNA targets and their introns. Structural studies of the dynamic interaction in a sequential modification system will be studied using lead (II) induced cleavage mapping and phosphorothioate-iodine footprinting. Functional characterization of these sRNP complexes will be done using thin layer chromatography of RNase T2 or nuclease P1 digests of their in vitro reaction products. In vivo cross-linking approaches will be utilized to isolate sRNP associated proteins. In vivo characterization of sRNP guide modifications and their relationship with splicing will be studied with a modified version of tRNATrp gene. The tRNA product of this modified gene and normal genomic gene can be distinguished. Products of various mutated versions of this modified gene will be characterized in in vivo studies. Role of the intron in wobble base modification of tRNA and its effect on the accuracy of translation will be tested by using a specifically modified reporter gene and a tRNATrp intron-deleted H. volcanii strain. Finally tRNA pseudouridine synthases, Pus10 and aCbf5 will be studied in vitro and in vivo for their ability to produce pseudouridine at positions 54 and/or 55 of tRNAs. Overall the basic understanding of these processes in archaeal systems will help us understand similar processes in human systems under normal conditions and the changes that may occur under diseased conditions. PUBLIC HEALTH RELEVANCE: Studies of gene regulatory phenomena such as RNA processing help us in understanding cellular metabolism and growth under normal as well as diseased conditions. Studying these processes in complex system is often challenging and sometimes not even feasible. Using simple model organisms, we elucidate the mechanisms and functions of these events, which can then be correlated to human systems.